Cytoplasmic male sterility gene ORF147 of pigeonpea, and uses thereof

ABSTRACT

The present disclosure provides, a DNA construct comprising a polynucleotide fragment comprising a first, and a second sequence, wherein said first sequence encodes a mitochondrial transit peptide, said second sequence encodes a polypeptide having amino acid sequence as set forth in SEQ ID NO: 1, and said polynucleotide fragment is operably linked to a flower specific promoter. The present disclosure also provides with a DNA vector, a recombinant host cell and a method of obtaining the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. § 371of International Application No. PCT/IN2017/050564, filedinternationally on Dec. 1, 2017, which claims priority to and thebenefit of Indian Application No. 201641041375, filed on Dec. 2, 2016,the disclosures of each of which are herein incorporated by reference intheir entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:586862000700SUBSEQLIST.TXT, date recorded: Jan. 27, 2020, size: 21 KB).

FIELD OF INVENTION

The present disclosure relates to the field of plant biology and plantbreeding. In particular, the disclosure provides an isolatedpolynucleotide fragment (cDNA) specific to pigeonpea that encodes apolypeptide, expression of which in mitochondria in floral specificmanner results in male sterility.

BACKGROUND OF THE INVENTION

The cytoplasmic male sterility (CMS) phenotype is an outcome ofincompatible interactions between the mitochondrial and nuclear genomes.The plant mitochondrial genome constitutes many sequences that activelyrecombine among themselves either by dividing into sub-genomic moleculesor by joining with other molecules to form new ones. These recombinationprocesses in mitochondrial genomes may create new molecular structuresand novel open reading frames (ORFs), including CMS genes (Mackenzie etal., Plant Cell, 1999, 11, 571-585).

Several mutations are associated with the CMS trait. They include, theT-urf13 gene in Zea mays (Dewey et al. Cell, 1986, 44, 439-449), pcfgene in Petunia (Young et al., Cell, 1987, 50, 41-49), cox1 in Oryzasativa (Wang et al. Plant Cell, 2006, 18, 676-687) and mutations inATPase subunits in Helianthus annuus (Laver et al., Plant J., 1991, 1,185-193) and Brassica napus (Landgren et al., Plant Mol. Bio., 1996, 32,879-890). The distinct variability in size and the multipartitestructures of plant mitochondrial genomes make it difficult to study thephysiological mechanism of CMS (Hanson et al., Plant Cell, 2004, 16,S154-169).

Pigeonpea [Cajanus cajan (L.) Millsp.] is an important high protein(20-22%) food legume of the rainfed tropics and sub-tropics of Asia,Africa and South America, cultivated by smallholder farmers. While theself-pollinating nature of legumes is a major bottleneck in exploitinghybrid vigor in these crops, pigeonpea has a unique advantage of beingpartially out-crossed (20 to 50%). Exploiting this, pigeonpea hybridtechnology with A₄ (C. cajanifolius) cytoplasm involving a three-parentsystem is considered one of the breakthrough technological interventionsin pulse breeding. This male sterile source designated as ICPA 2039, hasbeen transferred into a number of genetic backgrounds and is highlystable across environments (Saxena et al. Euphytica, 2005, 145, 289-294;Saxena et al., J. Hered., 2010, 101, 497-503), however, the source ofmale sterility remains unknown, which remains a limitation indevelopment of newer hybrid pigeonpea varieties for enhanced yield andquality. Elucidation can also potentially open the window forapplication and development of CMS lines in other pulses also.

SUMMARY OF THE INVENTION

In an aspect of the present disclosure, there is provided a DNAconstruct comprising a polynucleotide fragment, said polynucleotidefragment comprising a first, and a second sequence, wherein said firstsequence encodes a mitochondrial transit peptide, said second sequenceencodes a polypeptide having amino acid sequence as set forth in SEQ IDNO: 1, and said polynucleotide fragment is operably linked to a flowerspecific promoter.

In an aspect of the present disclosure, there is provided a DNA vectorcomprising a DNA construct, said DNA construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide, said second sequence encodes apolypeptide having amino acid sequence as set forth in SEQ ID NO: 1, andsaid polynucleotide fragment is operably linked to a promoter thatdrives the expression to floral organs.

In an aspect of the present disclosure, there is provided a recombinanthost cell comprising a DNA construct or a DNA vector comprising said DNAconstruct, said DNA construct comprising a polynucleotide fragment, saidpolynucleotide fragment comprising a first, and a second sequence,wherein said first sequence encodes a mitochondrial transit peptide,said second sequence encodes a polypeptide having amino acid sequence asset forth in SEQ ID NO: 1, and said polynucleotide fragment is operablylinked to promoter that drives the expression to floral organs.

In an aspect of the present disclosure, there is provided a male sterileplant harboring in its genome a DNA construct, said DNA constructcomprising a polynucleotide fragment, said polynucleotide fragmentcomprising a first, and a second sequence, wherein said first sequenceencodes a mitochondrial transit peptide, said second sequence encodes apolypeptide having amino acid sequence as set forth in SEQ ID NO: 1, andsaid polynucleotide fragment is operably linked to a promoter thatdrives the expression to floral organs.

In an aspect of the present disclosure, there is provided a method ofobtaining a male sterile plant harboring in its genome a DNA construct,said DNA construct comprising a polynucleotide fragment, saidpolynucleotide fragment comprising a first, and a second sequence,wherein said first sequence encodes a mitochondrial transit peptide,said second sequence encodes a polypeptide having amino acid sequence asset forth in SEQ ID NO: 1, and said polynucleotide fragment is operablylinked to promoter that drives the expression to floral organs, saidmethod comprising: (a) obtaining a DNA construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide, said second sequence encodes apolypeptide having amino acid sequence as set forth in SEQ ID NO: 1, andsaid polynucleotide fragment is operably linked to promoter that drivesthe expression to floral organs from the group consisting AP3 promoterfor floral expression from Arabidopsis (U30729), AP3 promoter for floralexpression from Tomato (DQ539418.1), AP3 Promoter from Coffea Arabica(AHW58030.1), TA29 Promoter for Tapetum-specific expression fromLycopersicon esculentum (AM261325.1), and TA29 Promoter from Tobacco forTapetum-specific expression (X52283) or a recombinant host cellcomprising a DNA vector comprising said DNA construct; (b) transformingplant cells with said DNA construct or recombinant host cell; and (c)selecting and developing a transgenic plant capable of heterologouslyexpressing a polypeptide having amino acid sequence as set forth in SEQID NO: 6, wherein said plant a male sterile plant.

In an aspect of the present disclosure, there is provided a method ofidentification of transgenic male sterile plants, said methodcomprising: (a) obtaining a transgenic plant biological materialcomprising DNA; (b) carrying out an amplification reaction using primerswhich bind to a DNA sequence as set forth in SEQ ID NO: 7 to generateamplicons; and (c) detecting the presence of said amplicons, whereinpresence of said amplicons is indicative of the transgenic plant beingmale sterile.

In an aspect of the present disclosure, there is provided an isolatedpolynucleotide fragment comprising a first, and a second sequence,wherein the first sequence encodes a mitochondrial transit peptide, andsaid second sequence encodes a polypeptide having amino acid sequence asset forth in SEQ ID NO: 1.

These and other features, aspects, and advantages of the present subjectmatter will be better understood with reference to the followingdescription and appended claims. This summary is provided to introduce aselection of concepts in a simplified form. This summary is not intendedto identify key features or essential features of the claimed subjectmatter, nor is it intended to be used to limit the scope of the claimedsubject matter.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

The following drawings form part of the present specification and areincluded to further illustrate aspects of the present disclosure. Thedisclosure may be better understood by reference to the drawings incombination with the detailed description of the specific embodimentspresented herein.

FIG. 1A depicts the RT-PCR analysis of nad7 in both male fertile, andsterile plants, in accordance with an embodiment of the presentdisclosure.

FIG. 1B depicts the amplification product of orf133 in parental linesand hybrid, in accordance with an embodiment of the present disclosure.

FIG. 1C depicts the amplification product of orf147 in CMS line and F1hybrid, in accordance with an embodiment of the present disclosure.

FIG. 2A depicts the organization of mitochondrial genomic regionsassociated with the orf147 gene in the male sterile pigeonpea line (ICPA2039), in accordance with an embodiment of the present disclosure.

FIG. 2B depicts the amplification of 1,327 bp (left) and 1,005 bp(right) regions spanning orf133, orf147 and part of the nad7 cds (codingDNA sequence) in male sterile line and hybrid, in accordance with anembodiment of the present disclosure.

FIG. 2C depicts the amplification of 2,143 bp (S1 & H1) and 1,741 bp (S2& H2) regions spanning the orf133, orf147 and nad7 cds in the malesterile line and hybrid, in accordance with an embodiment of the presentdisclosure.

FIG. 3A, B depicts the DNA sequencing profile used to identify thetranscription start sites (TSSs) in pigeonpea male sterile line, inaccordance with an embodiment of the present disclosure. SEQ ID NO: 47is shown in FIG. 3A; SEQ ID NO: 48 is shown in FIG. 3B.

FIG. 3C depicts the DNA sequencing profile used to identify thetranscription start sites (TSSs) in pigeonpea maintainer fertile line,in accordance with an embodiment of the present disclosure. SEQ ID NO:48 is shown.

FIG. 3D depicts the comparison of nucleotide sequences of the 5′flanking region of nad7 in male fertile (upper) and CMS (lower) lines,in accordance with an embodiment of the present disclosure. SEQ ID NO:49 is the top sequence and SEQ ID NO: 50 is the bottom sequence in thealignment.

FIG. 4A depicts the Secondary structure of the transcript predicted fromORF147 from the male sterile line using Sfold 2.2 software, inaccordance with an embodiment of the present disclosure. SEQ ID NO: 51is shown.

FIG. 4B depicts the predicted amino acid sequence of ORF147, inaccordance with an embodiment of the present disclosure. SEQ ID NO: 1 isshown.

FIG. 5A depicts the effect of expression of orf147 on E. coli growth, inaccordance with an embodiment of the present disclosure.

FIG. 5B depicts the effect of expression of orf133 on E. coli growth, inaccordance with an embodiment of the present disclosure.

FIG. 5C depicts the bacterial growth upon expression of orf147 ororf133, in accordance with an embodiment of the present disclosure.

FIG. 6A depicts the schematic representation of T-DNA region of a planttransformation vector carrying the candidate cytoplasmic male sterilitygene causing mitochondrial orf147 isolated from CMS pigeonpea line andfused with a yeast mitochondrial targeted peptide (CoxIV), cloned underthe AP3 promoter from Arabidopsis thaliana, in accordance with anembodiment of the present disclosure.

FIG. 6B depicts the male sterile transgenic Arabidopsis plant showingnormal growth and development, in accordance with an embodiment of thepresent disclosure.

FIG. 6C depicts the wild type plant with primary branches showing normalsiliques, in accordance with an embodiment of the present disclosure.

FIG. 6D depicts the male sterile transgenic plant with short siliquesindicating no developing seeds in accordance with an embodiment of thepresent disclosure.

FIG. 6E depicts the front view of normal mature flowers of WT (insetshows normal anther dehiscence), in accordance with an embodiment of thepresent disclosure.

FIG. 6F depicts the flowers of male sterile line revealing fusedcarpels, protruding pistil and short filaments (inset non-dehiscentanther in the transgenic flower), in accordance with an embodiment ofthe present disclosure.

FIG. 6G depicts the flower size, color and structure in the WT tobaccoplant, in accordance with an embodiment of the present disclosure.

FIG. 6H depicts the flowers of male sterile tobacco plants havinganthers below the stigma, in accordance with an embodiment of thepresent disclosure.

FIG. 6I depicts the seed capsules from N. tabacum (WT) (top), and thatof sterile progeny (bottom), in accordance with an embodiment of thepresent disclosure.

FIG. 6J depicts the seed capsules of WT plants (Left), collapsed anddetached seed capsules in partially sterile transgenic phenotypes(Inset) floral branches from wild type (WT), in accordance with anembodiment of the present disclosure.

FIG. 7A depicts the expression of mitochondrial orf147 in pigeonpea malesterile plant (ICPA 2039) vs. pigeonpea maintainer line (ICPB 2039), inaccordance with an embodiment of the present disclosure.

FIG. 7B depicts the expression of mitochondrial orf147 in completelysterile transgenic Arabidopsis (left), and partially sterile transgenictobacco (right), in a accordance with an embodiment of the presentdisclosure.

FIG. 8A depicts the real-time qRT-PCR showing reduced gene expression ofTDF1, DYT and MS1 in flower buds of the male sterile pigeonpea line(ICPA 2039) compared to those of the fertile maintainer line (ICPB2039), in accordance with an embodiment of the present disclosure.

FIG. 8B depicts the male sterile transgenic Arabidopsis showingdecreased expressions of key genes DYT1, AMS and MS1, in accordance withan embodiment of the present disclosure.

FIG. 9A depicts the phloroglucinol stained anthers in a bunch of flowersof wild type Arabidopsis plant, in accordance with an embodiment of thepresent disclosure.

FIG. 9B depicts the close up view of deeply stained (phloroglucinol) WTArabidopsis anther, in accordance with an embodiment of the presentdisclosure.

FIG. 9C depicts the anthers of a male sterile transgenic plant withreduced staining (phloroglucinol) indicating reduced lignification(inset), in accordance with an embodiment of the present disclosure.

FIG. 9D depicts the WT transgenic tobacco anthers accumulating stain(phloroglucinol), in accordance with an embodiment of the presentdisclosure.

FIG. 9E depicts the anthers of partially sterile tobacco transgenicplant, in accordance with an embodiment of the present disclosure.

FIG. 9F depicts the anthers of fully sterile male plant accumulatingvery little stain (phloroglucinol), in accordance with an embodiment ofthe present disclosure.

FIG. 9G depicts the reduced relative gene expression of ligninbiosynthesis genes such as 4CL (4 Coumarate:CoAligase), CCoAOMT(Caffeoyl CoA O-methyltransferase), and C3H (Cinnamic acid3-hydroxylase) in male sterile transgenic Arabidopsis plants, inaccordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

Those skilled in the art will be aware that the present disclosure issubject to variations and modifications other than those specificallydescribed. It is to be understood that the present disclosure includesall such variations and modifications. The disclosure also includes allsuch steps, features, compositions and compounds referred to orindicated in this specification, individually or collectively, and anyand all combinations of any or more of such steps or features.

Definitions

For convenience, before further description of the present disclosure,certain terms employed in the specification, and examples are collectedhere. These definitions should be read in the light of the remainder ofthe disclosure and understood as by a person of skill in the art. Theterms used herein have the meanings recognized and known to those ofskill in the art, however, for convenience and completeness, particularterms and their meanings are set forth below.

The articles “a”, “an” and “the” are used to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included. It is notintended to be construed as “consists of only”.

Throughout this specification, unless the context requires otherwise theword “comprise”, and variations such as “comprises” and “comprising”,will be understood to imply the inclusion of a stated element or step orgroup of element or steps but not the exclusion of any other element orstep or group of element or steps.

The term “including” is used to mean “including but not limited to”.“Including” and “including but not limited to” are used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the disclosure, the preferred methods, andmaterials are now described. All publications mentioned herein areincorporated herein by reference. The present disclosure is not to belimited in scope by the specific embodiments described herein, which areintended for the purposes of exemplification only.Functionally-equivalent products, compositions, and methods are clearlywithin the scope of the disclosure, as described herein.

Sequences:

SEQ ID NO: 1 depicts the amino acid sequence of polypeptide encoded byorf147.

MHLVLSFFPVCRSASKERKLKANKDKMTREIKLYVDTTPSDLDFMMNSDTDLQSLSSPDSSDAQSASPDLDLLWDQVCGEYHKCVHESGRVLPPEWTMPDLVRAVISDDEAIEQGFLTDAYYDVMLCGTHSWVCEELLNFLDLIHYG

SEQ ID NO: 2 depicts the mitochondrial transit peptide amino acidsequence.

MLSLRQSIRFFKPATRTLCSSRYLLQQKP

SEQ ID NO: 3 depicts the nucleotide sequence of orf147.

ATGCATCTGGTTCTATCTTTTTTTCCGGTATGCCGCTCCGCCAGCAAGGAGCGAAAACTAAAAGCAAACAAAGATAAGATGACCAGAGAGATCAAGCTATATGTGGATACCACCCCTAGTGATTTGGATTTTATGATGAATAGTGATACGGATTTGCAGTCCTTGTCTTCCCCGGATTCGTCCGACGCACAGAGTGCTTCACCGGACTTGGACCTATTATGGGATCAAGTTTGTGGTGAATACCACAAGTGTGTGCATGAATCCGGGAGGGTCTTACCCCCGGAATGGACGATGCCCGACCTTGTTCGGGCTGTTATTTCCGACGATGAAGCTATTGAGCAGGGCTTTCTGACGGATGCCTACTATGATGTCATGTTATGTGGCACTCATAGTTGGGTATGCGAGGAGCTGCTTAATTTCCTCGATCTAATCCACTATGGCTGA

SEQ ID NO: 4 depicts the nucleotide sequence encoding mitochondrialtransit peptide.

ATGTTGTCACTACGTCAATCTATAAGATTTTTCAAGCCAGCCACAAGAACTTTGTGTAGCTCTAGATATCTGCTTCAGCAAAAACCC

SEQ ID NO: 5 depicts the Arabidopsis promoter sequence.

CAGTAACTGTGGCCAACTTAGTTTTGAAACAACACTAACTGGTCGAAGCAAAAAGAAAAAAGAGTTTCATCATATATCTGATTTGATGGACTGTTTGGAGTTAGGACCAAACATTATCTACAAACAAAGACTTTTCTCCTAACTTGTGATTCCTTCTTAAACCCTAGGGGTAATATTCTATTTTCCAAGGATCTTTAGTTAAAGGCAAATCCGGGAAATTATTGTAATCATTTGGGGAAACATATAAAAGATTTGAGTTAGATGGAAGTGACGATTAATCCAAACATATATATCTCTTTCTTCTTATTTCCCAAATTAACAGACAAAAGTAGAATATTGGCTTTTAACACCAATATAAAAACTTGTTCACACCTAAACACTTTTGTTTACTTTAGGGTAAGTGTAAAAAGCCAACCAAATCCACCTGCACTGATTTGACGTTTACAAACGCCGTTAAGTTTGTCACCGTCTAAACAAAAACAAAGTAGAAGCTAACGGAGCTCCGTTAATAAATTGACGAAAAGCAAACCAAGTTTTTAGCTTTGGTCCCCCTCTTTTACCAAGTGACAATTGATTTAAGCAGTGTCTTGTAATTATACAACCATCGATGTCCGTTGATTTAAACAGTGTCTTGTAATTAAAAAAATCAGTTTACATAAATGGAAAATTTATCACTTAGTTTTCATCAACTTCTGAACTTACCTTTCATGGATTAGGCAATACTTTCCATTTTTAGTAACTCAAGTGGACCCTTTACTTCTTCAACTCCATCTCTCTCTTTCTATTTCACTTCTTTCTTCTCATTATATCTCTTGTCCTCTCCACCAAATCTCTTCAACAAAAAGATTAAACAAAGAGAGAAGAATC AT

SEQ ID NO: 6 depicts the amino acid sequence of a polypeptide comprisingmitochondrial transit peptide fused to Orf147.

MLSLRQSIRFFKPATRTLCSSRYLLQQKPMHLVLSFFPVCRSASKERKLKANKDKMTREIKLYVDTTPSDLDFMMNSDTDLQSLSFPDSSDAQSASPDLDLLWDRVCGEYHKCVHESGRVLPPEWTMPDLVRAVISDDEAIEQGFLTDAYYDVMLCGTHSWVCEELLNFLDLIHYG

SEQ ID NO: 7 depicts the nucleotide sequence of a polynucleotidefragment encoding a mitochondrial transit peptide fused to orf147.

ATGTTGTCACTACGTCAATCTATAAGATTTTTCAAGCCAGCCACAAGAACTTTGTGTAGCTCTAGATATCTGCTTCAGCAAAAACCCATGCATCTGGTTCTATCTTTTTTTCCGGTATGCCGCTCCGCCAGCAAGGAGCGAAAACTAAAAGCAAACAAAGATAAGATGACCAGAGAGATCAAGCTATATGTGGATACCACCCCTAGTGATTTGGATTTTATGATGAATAGTGATACGGATTTGCAGTCCTTGTCTTTCCCGGATTCGTCTGACGCACAGAGTGCTTCACCGGACTTGGACCTATTATGGGATCGAGTTTGTGGTGAATACCACAAGTGTGTGCATGAATCCGGGAGGGTCTTACCCCCGGAATGGACGATGCCCGACCTTGTTCGGGCTGTTATTTCCGACGATGAAGCTATTGAGCAGGGCTTTCTGACGGATGCCTACTATGATGTCATGTTATGTGGCACTCATAGTTGGGTATGCGAGGAGCTGCTTAATTTCCTCGATCTAATCCACTATGGCTGA

SEQ ID NO: 8 depicts the antisense primer sequence used for PCR baseddirectional genome walking.

AATTCAAAGTGAAATTTTTG

SEQ ID NO: 9 depicts the forward primer sequence for amplifying orf133.

ATGCAGTTACTCTTTGAGTTGGA

SEQ ID NO: 10 depicts the reverse primer sequence for amplifying orf133.

TCATGCTCTTAACTTACCTTCTG

SEQ ID NO: 11 depicts the forward primer sequence for amplifying orf147.

ATGCATCTGGTTCTATCTT

SEQ ID NO: 12 depicts the reverse primer sequence for amplifying orf147.

TCAGCCATAGTGGATTAGATCGAG

SEQ ID NO: 13 depicts the forward primer sequence with Ndel restrictionsite for orf133.

TCAGCCATAGTGGATTAGATCGAG

SEQ ID NO: 14 depicts the reverse primer sequence with SalI restrictionsite for orf133.

ATAGTCGACTCATGCTCTTAACTTACCTTCTG

SEQ ID NO: 15 depicts the forward primer sequence with Ndel restrictionsite for orf147.

TATCATATGCATCTGGTTCTATCTT

SEQ ID NO: 16 depicts the forward primer sequence with SalI restrictionsite for orf147.

TATGTCGACTCAGCCATAGTGGATTAGATCG

SEQ ID NO: 17 depicts the forward primer with KpnI site for amplifyingAtAP3 promoter.

TAGGTACCCAGTAACTGTGGCCAACTTAGTT

SEQ ID NO: 18 depicts the reverse primer with NdeI site for amplifyingAtAP3 promoter.

TCAGATCATATGATTCTTCTCTCTTTGTTTAATCT

SEQ ID NO: 19 depicts the forward primer for amplifying CoxIVmitochondrial signal peptide.

ATGTTGTCACTACGTCAATCTATAAG

SEQ ID NO: 20 depicts the reverse primer for amplifying CoxIVmitochondrial signal peptide.

GGGTTTTTGCTGAAGCAGAT

SEQ ID NO: 21 depicts the forward primer for amplifying CoxIV orf147fusion.

ATCTGCTTCAGCAAAAACCCATGCATCTGGTTCTATCTTTTTTTC

SEQ ID NO: 22 depicts the reverse primer for amplifying CoxIV orf147fusion.

GAAAAAAAGATAGAACCAGATGCATGGGTTTTTGCTGAAGCAGAT

SEQ ID NO: 23 depicts the forward primer with NdeI restriction site foramplifying CoxIV orf147 fusion.

TATCATATGTTGTCACTACGTCAATCTATAAG

SEQ ID NO: 24 depicts the reverse primer with NotI restriction site foramplifying CoxIV orf147 fusion.

GCGGCCGCTCAGCCATAGTGGATTAGATCG

SEQ ID NO: 25 depicts the forward primer sequence for amplifying DYT1.

GAAGCTCCTCCTGAGATTGATG

SEQ ID NO: 26 depicts the reverse primer sequence for amplifying DYT1.

CTTCCTCTCCCCAATCTTACAC

SEQ ID NO: 27 depicts the forward primer sequence for amplifying AMS.

AGGCTCTATGCAAAACGAAAAG

SEQ ID NO: 28 depicts the reverse primer sequence for amplifying AMS.

GGTTGTGGTAATGGTTGATGTG

SEQ ID NO: 29 depicts the forward primer sequence for amplifying SAND.

GTGCAGACACAAGGTTGTCAGT

SEQ ID NO: 30 depicts the reverse primer sequence for amplifying SAND.

GGTAGGCAGATTGGTGAGAAAG

SEQ ID NO: 31 depicts the forward primer sequence for amplifying TIP41.

GAAGATGAGGCACCAACTGTTC

SEQ ID NO: 32 depicts the reverse primer sequence for amplifying TIP41.

GCTTAATCACTGGAAGCCTCTG

SEQ ID NO: 33 depicts the forward primer sequence for amplifying UNK.

GCTGAGAAGCATGTTCAGGAGT

SEQ ID NP: 34 depicts the reverse primer sequence for amplifying UNK.

GTTCATGAGCTCAGAGAGACCA

SEQ ID NO: 35 depicts the forward primer sequence for amplifying C3H.

AGTTCGACAGAGTGGTTGGACT

SEQ ID NO: 36 depicts the reverse primer sequence for amplifying C3H.

GCTTCGGTGAGGTAGCATTAGA

SEQ ID NO: 37 depicts the forward primer sequence for amplifyingCCoAOMT.

CTGGCTATGGATGTCAACAGAG

SEQ ID NO: 38 depicts the reverse primer sequence for amplifyingCCoAOMT.

GTTCCATGGTTCTTCTCGTCAG

SEQ ID NO: 39 depicts the forward primer sequence for amplifying 4CL.

AGGAACCTTTTCCGGTTAAGTC

SEQ ID NO: 40 depicts the reverse primer sequence for amplifying 4CL.

GATCTGGTGACCACGAATACAA

SEQ ID NO: 41 depicts Nad7pGER:

CTATCCACCTCTCCAGACAC

SEQ ID NO: 42 depicts Nad7p1R:

CAAAAATTTCACTTCGAATT

SEQ ID NO: 43 depicts RACEF1:

ATGACGACTAGGAACGGGCAAATC

SEQ ID NO: 44 depicts RACE R1:

GATCGAGGAAATTAAGCAGCTC

SEQ ID NO: 45 depicts RACE R2:

CCCGACAGAGAGGGAAAAG

SEQ ID NO: 46 depicts orf 133

ATGCAGTTACTCTTTGAGTTGGAGTCTCTTCCGGGGTTTTGCTCCAGCCTGTCCCTTACAGATCCAGTAGTTGCAGTTCCCCCTTTTCTGTCGTCGGAAAGAGGCTCTAACAAGACCTCGAACCAGCACCTCTGGGAGAAAAAGCGAACTGCCCTTCGTTCTTATCGTGACAAAAGTTCTGTTAGGTTCTTAGTAGCAATCGGCGACCTTTTCCTTCTTCTTTCCCTTTTCCCTCTCTGTCGGGGTCAGGGGTGTCTTCCCTTTGCTGGCCATCTTGGAAGAAATAGTTTAGGAAGGTCAGGGATACGATTTGAATCCCGAGCCCCGATAGCCAATCCGAATAAAGCGGCAGATCTGCGGACTCAGGAAAGAAAGCTTTCAGAAGGTAAGTTAAGAGCAT GA

In an embodiment of the present disclosure, there is provided a DNAconstruct comprising a polynucleotide fragment, said polynucleotidefragment comprising a first, and a second sequence, wherein said firstsequence encodes a mitochondrial transit peptide, said second sequenceencodes a polypeptide having amino acid sequence as set forth in SEQ IDNO: 1, and said polynucleotide fragment is operably linked to promoterthat drives the expression to floral organs. The floral promoter isselected from the group consisting of AP3 promoter for floral expressionfrom Arabidopsis (U30729), AP3 promoter for floral expression fromTomato (DQ539418.1), AP3 Promoter from Coffea Arabica (AHW58030.1), TA29Promoter for Tapetum-specific expression from Lycopersicon esculentum(AM261325.1), and TA29 Promoter from Tobacco for Tapetum-specificexpression (X52283).

In an embodiment of the present disclosure, there is provided a DNAconstruct as described herein, wherein said second sequence is as setforth in SEQ ID NO: 3. In an embodiment of the present disclosure, thereis provided a DNA construct as described herein, wherein saidmitochondrial transit peptide is selected from the group consisting ofmitochondrial transit peptide of the cytochrome oxidase subunit IV fromyeast, and COX4 from Saccharomyces cerevisiae (P04037|1-25).

In an embodiment of the present disclosure, there is provided a DNAconstruct as described herein, wherein said mitochondrial transitpeptide amino acid sequence is as set forth in SEQ ID NO: 2, and isfused in-frame at the 5′ end of said second sequence.

In an embodiment of the present disclosure, there is provided a DNAconstruct as described herein, wherein said mitochondrial transitpeptide amino acid sequence is as set forth in SEQ ID NO: 2, and isfused in-frame at the 5′ end of SEQ ID NO: 1.

In an embodiment of the present disclosure, there is provided a DNAconstruct as described herein, wherein said first sequence is as setforth in SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided a DNAconstruct as described herein, wherein said flower specific promoter isselected from the group consisting of AP3 promoter for floral expressionfrom Arabidopsis (U30729), AP3 promoter for floral expression fromTomato (DQ539418.1), AP3 Promoter from Coffea Arabica (AHW58030.1), TA29Promoter for Tapetum-specific expression from Lycopersicon esculentum(AM261325.1), and TA29 Promoter from Tobacco for Tapetum-specificexpression (X52283). The developmentally regulated APETALA3 promoterfrom Arabidopsis thaliana (AP3) which is specific to petals and stamens,(Jack, T. et al. (1994) Cell 76, 703-716) AP3 was chosen as a tissuespecific promoter because it is expressed very early in the developmentof the stamen (as well as the petal).). A person skilled in the art canuse any flower or anther specific promoter to drive the expression oforf147 fragment

In an embodiment of the present disclosure, there is provided a DNAconstruct as described herein, wherein said flower specific promotersequence is as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a DNAconstruct comprising a polynucleotide fragment, said polynucleotidefragment comprising a first, and a second sequence, wherein said firstsequence encodes a mitochondrial transit peptide having amino acidsequence as set forth in SEQ ID NO: 2, and said second sequence encodesa polypeptide having amino acid sequence as set forth in SEQ ID NO: 1,and said polynucleotide fragment is operably linked to a flower specificpromoter having sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a DNAconstruct comprising a polynucleotide fragment, said polynucleotidefragment comprising a first, and a second sequence, wherein said firstsequence encodes a mitochondrial transit peptide encoded by a sequenceas set forth in SEQ ID NO: 4, and said second sequence is as set forthin SEQ ID NO: 3, and said polynucleotide fragment is operably linked toa flower specific promoter having sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a DNAvector comprising a DNA construct, said DNA construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide, said second sequence encodes apolypeptide having amino acid sequence as set forth in SEQ ID NO: 1, andsaid polynucleotide fragment is operably linked to a flower specificpromoter.

In an embodiment of the present disclosure, there is provided a DNAvector as described herein, wherein said second sequence is as set forthin SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided a DNAvector as described herein, wherein said mitochondrial transit peptideis selected from the group consisting of mitochondrial transit peptideof the cytochrome oxidase subunit IV from yeast, and COX4 fromSaccharomyces cerevisiae (P04037|1-25). A person skilled in the art canuse any other mitochondrial transit peptide.

In an embodiment of the present disclosure, there is provided a DNAvector as described herein, wherein said mitochondrial transit peptideamino acid sequence is as set forth in SEQ ID NO: 2, and is fusedin-frame at the 5′ end of said second sequence.

In an embodiment of the present disclosure, there is provided a DNAvector as described herein, wherein said mitochondrial transit peptideamino acid sequence is as set forth in SEQ ID NO: 2, and is fusedin-frame at the 5′ end of SEQ ID NO: 1.

In an embodiment of the present disclosure, there is provided a DNAvector as described herein, wherein said first sequence is as set forthin SEQ ID NO: 4. In an embodiment of the present disclosure, there isprovided a DNA vector as described herein, wherein said flower specificpromoter is selected from the group consisting of AP3 promoter forfloral expression from Arabidopsis (U30729), AP3 promoter for floralexpression from Tomato (DQ539418.1), AP3 Promoter from Coffea Arabica(AHW58030.1), TA29 Promoter for Tapetum-specific expression fromLycopersicon esculentum (AM261325.1), and TA29 Promoter from Tobacco forTapetum-specific expression (X52283). The developmentally regulatedAPETALA3 promoter from Arabidopsis thaliana (AP3) which is specific topetals and stamens, (Jack, T. et al. (1994) Cell 76, 703-716); AP3 waschosen as a tissue specific promoter because it is expressed very earlyin the development of the stamen (as well as the petal).

In an embodiment of the present disclosure, there is provided a DNAvector as described herein, wherein said anther specific promotersequence is as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a DNAvector comprising a DNA construct, wherein said DNA construct comprisesa polynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide having amino acid sequence as set forth inSEQ ID NO: 2, and said second sequence encodes a polypeptide havingamino acid sequence as set forth in SEQ ID NO: 1, and saidpolynucleotide fragment is operably linked to a flower specific promoterhaving sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a DNAvector comprising a DNA construct, wherein said DNA construct comprisesa polynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide encoded by a sequence as set forth in SEQID NO: 4, and said second sequence as set forth in SEQ ID NO: 3, andsaid polynucleotide fragment is operably linked to a flower specificpromoter having sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct, said DNA constructcomprising a polynucleotide fragment, said polynucleotide fragmentcomprising a first, and a second sequence, wherein said first sequenceencodes a mitochondrial transit peptide, said second sequence encodes apolypeptide having amino acid sequence as set forth in SEQ ID NO: 1, andsaid polynucleotide fragment is operably linked to a flower specificpromoter.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said second sequence is as set forth in SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said mitochondrial transit peptide is selected from the groupconsisting of mitochondrial transit peptide of the cytochrome oxidasesubunit IV from yeast, and COX4 from Saccharomyces cerevisiae(P04037|1-25).

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said mitochondrial transit peptide amino acid sequence is as setforth in SEQ ID NO: 2, and is fused in-frame at the 5′ end of saidsecond sequence.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said mitochondrial transit peptide amino acid sequence is as setforth in SEQ ID NO: 2, and is fused in-frame at the 5′ end of SEQ ID NO:1.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said first sequence is as set forth in SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said flower specific promoter is selected from the groupconsisting of AP3 promoter for floral expression from Arabidopsis(U30729), AP3 promoter for floral expression from Tomato (DQ539418.1),AP3 Promoter from Coffea Arabica (AHW58030.1), TA29 Promoter forTapetum-specific expression from Lycopersicon esculentum (AM261325.1),and TA29 Promoter from Tobacco for Tapetum-specific expression (X52283).The developmentally regulated APETALA3 promoter from Arabidopsisthaliana (AP3) which is specific to petals and stamens, (Jack, T. et al.(1994) Cell 76, 703-716); AP3 was chosen as a tissue specific promoterbecause it is expressed very early in the development of the stamen (aswell as the petal).

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said flower specific promoter sequence is as set forth in SEQ IDNO: 5.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct, said DNA constructcomprising a polynucleotide fragment, said polynucleotide fragmentcomprising a first, and a second sequence, wherein said first sequenceencodes a mitochondrial transit peptide having amino acid sequence asset forth in SEQ ID NO: 2, and said second sequence encodes apolypeptide having amino acid sequence as set forth in SEQ ID NO: 1, andsaid polynucleotide fragment is operably linked to a flower specificpromoter having sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct, said DNA constructcomprising a polynucleotide fragment, said polynucleotide fragmentcomprising a first, and a second sequence, wherein said first sequenceencodes a mitochondrial transit peptide encoded by a sequence as setforth in SEQ ID NO: 4, and said second sequence as set forth in SEQ IDNO: 3, and said polynucleotide fragment is operably linked to a flowerspecific promoter having sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said recombinant host cell is a plant cell.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said recombinant host cell is a monocot.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said recombinant host cell is a dicot.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said recombinant host cell is selected from the group consistingof kidney bean, lima bean, mung bean, black gram, broad bean, fieldbean, garden pea, chick pea, black eyed pea, pigeonpea, tobacco, rice,maize, wheat, sorghum, and lentil.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said recombinant host cell is tobacco or Arabidopsis.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA construct as described herein,wherein said recombinant host cell is pigeonpea or lentil.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector, said DNA vectorcomprising a DNA construct, said DAN construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide, said second sequence encodes apolypeptide having amino acid sequence as set forth in SEQ ID NO: 1, andsaid polynucleotide fragment is operably linked to a flower specificpromoter.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said second sequence is as set forth in SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said mitochondrial transit peptide is selected from the groupconsisting of mitochondrial transit peptide of the cytochrome oxidasesubunit IV from yeast, and COX4 from Saccharomyces cerevisiae(P04037|1-25).

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said mitochondrial transit peptide amino acid sequence is as setforth in SEQ ID NO: 2, and is fused in-frame at the 5′ end of saidsecond sequence.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said mitochondrial transit peptide amino acid sequence is as setforth in SEQ ID NO: 2, and is fused in-frame at the 5′ end of SEQ ID NO:1.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said first sequence is as set forth in SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said flower specific promoter is selected from the groupconsisting of AP3 promoter for floral expression from Arabidopsis(U30729), AP3 promoter for floral expression from Tomato (DQ539418.1),AP3 Promoter from Coffea Arabica (AHW58030.1), TA29 Promoter forTapetum-specific expression from Lycopersicon esculentum (AM261325.1),and TA29 Promoter from Tobacco for Tapetum-specific expression (X52283).The developmentally regulated APETALA3 promoter from Arabidopsisthaliana (AP3) which is specific to petals and stamens, (Jack, T. et al.(1994) Cell 76, 703-716); AP3 was chosen as a tissue specific promoterbecause it is expressed very early in the development of the stamen (aswell as the petal).

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said anther specific promoter sequence is as set forth in SEQ IDNO: 5.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector, said DNA vectorcomprising a DNA construct, said DNA construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide having amino acid sequence as set forth inSEQ ID NO: 2, and said second sequence encodes a polypeptide havingamino acid sequence as set forth in SEQ ID NO: 1, and saidpolynucleotide fragment is operably linked to a flower specific promoterhaving sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector, said DNA vectorcomprising a DNA construct, said DNA construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide encoded by a sequence as set forth in SEQID NO: 4, and said second sequence as set forth in SEQ ID NO: 3, andsaid polynucleotide fragment is operably linked to a flower specificpromoter having sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said recombinant host cell is a fungal cell.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said recombinant host cell is a bacterial cell.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said recombinant host cell is Agrobacterium.

In an embodiment of the present disclosure, there is provided arecombinant host cell comprising a DNA vector as described herein,wherein said recombinant host cell is Agrobacterium tumefaciens.

In an embodiment of the present disclosure, there is provided a malesterile plant harboring in its genome a DNA construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide, said second sequence encodes apolypeptide having amino acid sequence as set forth in SEQ ID NO: 1, andsaid polynucleotide fragment is operably linked to a flower or antherspecific promoter.

In an embodiment of the present disclosure, there is provided a malesterile plant as described herein, wherein said second sequence is asset forth in SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided a malesterile plant as described herein, wherein said mitochondrial transitpeptide is selected from the group consisting of mitochondrial transitpeptide of the cytochrome oxidase subunit IV from yeast, and COX4 fromSaccharomyces cerevisiae (P04037|1-25).

In an embodiment of the present disclosure, there is provided a malesterile plant as described herein, wherein said mitochondrial transitpeptide amino acid sequence is as set forth in SEQ ID NO: 2, and isfused in-frame at the 5′ end of said second sequence.

In an embodiment of the present disclosure, there is provided a malesterile plant as described herein, wherein said mitochondrial transitpeptide amino acid sequence is as set forth in SEQ ID NO: 2, and isfused in-frame at the 5′ end of SEQ ID NO: 1.

In an embodiment of the present disclosure, there is provided a malesterile plant as described herein, wherein said first sequence is as setforth in SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided a malesterile plant as described herein, wherein said flower specific promoteris selected from the group consisting of AP3 promoter for floralexpression from Arabidopsis (U30729), AP3 promoter for floral expressionfrom Tomato (DQ539418.1), AP3 Promoter from Coffea Arabica (AHW58030.1),TA29 Promoter for Tapetum-specific expression from Lycopersiconesculentum (AM261325.1), and TA29 Promoter from Tobacco forTapetum-specific expression (X52283). The developmentally regulatedAPETALA3 promoter from Arabidopsis thaliana (AP3) which is specific topetals and stamens, (Jack, T. et al. (1994) Cell 76, 703-716); AP3 waschosen as a tissue specific promoter because it is expressed very earlyin the development of the stamen (as well as the petal).

In an embodiment of the present disclosure, there is provided a malesterile plant as described herein, wherein said flower specific promotersequence is as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a malesterile plant harboring in its genome a DNA construct, said DNAconstruct comprising a polynucleotide fragment, said polynucleotidefragment comprising a first, and a second sequence, wherein said firstsequence encodes a mitochondrial transit peptide having amino acidsequence as set forth in SEQ ID NO: 2, and said second sequence encodesa polypeptide having amino acid sequence as set forth in SEQ ID NO: 1,and said polynucleotide fragment is operably linked to an flowerspecific promoter having sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a malesterile plant harboring in its genome a DNA construct, said DNAconstruct comprising a polynucleotide fragment, said polynucleotidefragment comprising a first, and a second sequence, wherein said firstsequence encodes a mitochondrial transit peptide encoded by a sequenceas set forth in SEQ ID NO: 4, and said second sequence as set forth inSEQ ID NO: 3, and said polynucleotide fragment is operably linked to anflower specific promoter having sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a malesterile plant as described herein, wherein said plant is a monocot.

In an embodiment of the present disclosure, there is provided a malesterile plant, wherein said plant is a dicot.

In an embodiment of the present disclosure, there is provided a malesterile plant, wherein said plant is selected from the group consistingof kidney bean, lima bean, mung bean, black gram, broad bean, fieldbean, garden pea, chickpea, black eyed pea, pigeonpea, tobacco, rice,maize, wheat, sorghum, and lentil.

In an embodiment of the present disclosure, there is provided a malesterile plant, wherein said plant is Arabidopsis.

In an embodiment of the present disclosure, there is provided a malesterile plant, wherein said plant is tobacco or pigeonpea.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant, said method comprising: (a) obtaininga DNA construct comprising a first, and a second sequence, wherein saidfirst sequence encodes a mitochondrial transit peptide, said secondsequence encodes a polypeptide having amino acid sequence as set forthin SEQ ID NO: 1, and said polynucleotide fragment is operably linked toan flower specific promoter; or a recombinant host cell comprising a DNAvector, said DNA vector comprising said DNA construct; (b) transformingplant cells with said DNA construct or recombinant host cell; and (c)selecting and developing a transgenic plant capable of heterologouslyexpressing a polypeptide having amino acid sequence as set forth in SEQID NO: 6, wherein said plant is a male sterile plant.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, wherein saidrecombinant host cell is Agrobacterium.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, whereintransformation is carried out by a method selected from the groupconsisting of Agrobacterium mediated transformation method, particle gunbombardment method, in-planta transformation method, liposome mediatedtransformation method, protoplast transformation method, microinjection,and macroinjection.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, whereintransformation is carried out by Agrobacterium mediated transformationmethod.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, whereintransformation is carried out by particle gun bombardment method.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, wherein saidsecond sequence is as set forth in SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, wherein saidmitochondrial transit peptide is selected from the group consisting ofmitochondrial transit peptide of the cytochrome oxidase subunit IV fromyeast, and COX4 from Saccharomyces cererisiae (P04037|1-25).

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, wherein saidmitochondrial transit peptide amino acid sequence is as set forth in SEQID NO: 2, and is fused in-frame at the 5′ end of said second sequence.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, wherein saidmitochondrial transit peptide amino acid sequence is as set forth in SEQID NO: 2, and is fused in-frame at the 5′ end of SEQ ID NO: 1.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, wherein saidfirst sequence is as set forth in SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, wherein saidflower specific promoter is selected from the group consisting of AP3promoter for floral expression from Arabidopsis (U30729), AP3 promoterfor floral expression from Tomato (DQ539418.1), AP3 Promoter from CoffeaArabica (AHW58030.1), TA29 Promoter for Tapetum-specific expression fromLycopersicon esculentum (AM261325.1), and TA29 Promoter from Tobacco forTapetum-specific expression (X52283). The developmentally regulatedAPETALA3 promoter from Arabidopsis thaliana (AP3) which is specific topetals and stamens, (Jack, T. et al. (1994) Cell 76, 703-716); AP3 waschosen as a tissue specific promoter because it is expressed very earlyin the development of the stamen (as well as the petal).

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile plant as described herein, wherein saidflower specific promoter sequence is as set forth in SEQ ID NO: 5.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile Arabidopsis plant, said method comprising:(a) obtaining a DNA construct comprising a polynucleotide fragment, saidpolynucleotide fragment comprising a first, and a second sequence,wherein said first sequence encodes a mitochondrial transit peptidehaving amino acid sequence as set forth in SEQ ID NO: 2, and said secondsequence encodes a polypeptide having amino acid sequence as set forthin SEQ ID NO: 1, and said polynucleotide fragment is operably linked toa flower specific promoter having sequence as set forth in SEQ ID NO: 5;(b) transforming Arabidopsis cells with said DNA construct by particlegun bombardment method; and (c) selecting and developing a transgenicArabidopsis plant capable of heterologously expressing a polypeptidehaving amino acid sequence as set forth in SEQ ID NO: 6, wherein saidArabidopsis plant is a cytoplasmic male sterile Arabidopsis plant.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile tobacco plant, said method comprising: (a)obtaining a DNA construct comprising a polynucleotide fragment, saidpolynucleotide fragment comprising a first, and a second sequence,wherein said first sequence encodes a mitochondrial transit peptidehaving amino acid sequence as set forth in SEQ ID NO: 2, and said secondsequence encodes a polypeptide having amino acid sequence as set forthin SEQ ID NO: 1, and said polynucleotide fragment is operably linked toa flower specific promoter having sequence as set forth in SEQ ID NO: 5;(b) transforming tobacco cells with said DNA construct by particle gunbombardment method; and (c) selecting and developing a transgenictobacco plant capable of heterologously expressing a polypeptide havingamino acid sequence as set forth in SEQ ID NO: 6, wherein said tobaccoplant is a male sterile tobacco plant.

In an embodiment of the present disclosure, there is provided a methodof obtaining a cytoplasmic male sterile pigeonpea plant, said methodcomprising: (a) obtaining a DNA construct comprising a polynucleotidefragment, said polynucleotide fragment comprising a polynucleotidefragment, said polynucleotide fragment comprising a first, and a secondsequence, wherein said first sequence encodes a mitochondrial transitpeptide encoded by a sequence as set forth in SEQ ID NO: 4, and saidsecond sequence as set forth in SEQ ID NO: 3, and said polynucleotidefragment is operably linked to a flower specific promoter havingsequence as set forth in SEQ ID NO: 5; (b) transforming pigeonpea cellswith said DNA construct by particle gun bombardment method; and (c)selecting and developing a transgenic pigeonpea plant capable ofheterologously expressing a polypeptide having amino acid sequence asset forth in SEQ ID NO: 6, wherein said pigeonpea plant is a cytoplasmicmale sterile pigeonpea plant.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile tobacco plant, said method comprising: (a)obtaining a DNA construct comprising a polynucleotide fragment, saidpolynucleotide fragment comprising a first, and a second sequence,wherein said first sequence encodes a mitochondrial transit peptideencoded by a sequence as set forth in SEQ ID NO: 4, and said secondsequence as set forth in SEQ ID NO: 3, and said polynucleotide fragmentis operably linked to a flower specific promoter having sequence as setforth in SEQ ID NO: 5; (b) transforming tobacco cells with said DNAconstruct by particle gun bombardment method; and (c) selecting anddeveloping a transgenic tobacco plant capable of heterologouslyexpressing a polypeptide having amino acid sequence as set forth in SEQID NO: 6, wherein said tobacco plant is a male sterile tobacco plant.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile Arabidopsis plant, said method comprising:(a) obtaining an Agrobacterium host cell comprising a DNA vector, saidDNA vector comprising a DNA construct, said DNA construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide having amino acid sequence as set forth inSEQ ID NO: 2, and said second sequence encodes a polypeptide havingamino acid sequence as set forth in SEQ ID NO: 1, and saidpolynucleotide fragment is operably linked to a flower specific promoterhaving sequence as set forth in SEQ ID NO: 5; (b) transformingArabidopsis cells with said host cell by Agrobacterium mediatedtransformation method; and (c) selecting and developing a transgenicpigeonpea plant capable of heterologously expressing a polypeptidehaving amino acid sequence as set forth in SEQ ID NO: 6, wherein saidArabidopsis plant is a cytoplasmic male sterile Arabidopsis plant.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile tobacco plant, said method comprising: (a)obtaining an Agrobacterium host cell comprising a DNA vector, said DNAvector comprising a DNA construct, said DNA construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide having amino acid sequence as set forth inSEQ ID NO: 2, and said second sequence encodes a polypeptide havingamino acid sequence as set forth in SEQ ID NO: 1, and saidpolynucleotide fragment is operably linked to a flower specific promoterhaving sequence as set forth in SEQ ID NO: 5; (b) transforming tobaccocells with said host cell by Agrobacterium mediated transformationmethod; and (c) selecting and developing a transgenic tobacco plantcapable of heterologously expressing a polypeptide having amino acidsequence as set forth in SEQ ID NO: 6, wherein said tobacco plant is amale sterile tobacco plant.

In an embodiment of the present disclosure, there is provided a methodof obtaining a cytoplasmic male sterile Arabidopsis plant, said methodcomprising: (a) obtaining an Agrobacterium host cell comprising a DNAvector, said DNA vector comprising a DNA construct, said DNA constructcomprising a polynucleotide fragment, said polynucleotide fragmentcomprising a first, and a second sequence, wherein said first sequenceencodes a mitochondrial transit peptide having amino acid sequence asset forth in SEQ ID NO: 2, and said second sequence encodes apolypeptide having amino acid sequence as set forth in SEQ ID NO: 1, andsaid polynucleotide fragment is operably linked to a flower specificpromoter having sequence as set forth in SEQ ID NO: 5; (b) transformingArabidopsis cells with said host cell by Agrobacterium mediatedtransformation method; and (c) selecting and developing a transgenicArabidopsis plant capable of heterologously expressing a polypeptidehaving amino acid sequence as set forth in SEQ ID NO: 6, wherein saidpigeonpea plant is a cytoplasmic male sterile pigeonpea plant.

In an embodiment of the present disclosure, there is provided a methodof obtaining a male sterile tobacco plant, said method comprising: (a)obtaining an Agrobacterium host cell comprising a DNA vector, said DNAvector comprising a DNA construct, said DNA construct comprising apolynucleotide fragment, said polynucleotide fragment comprising afirst, and a second sequence, wherein said first sequence encodes amitochondrial transit peptide having amino acid sequence as set forth inSEQ ID NO: 2, and said second sequence encodes a polypeptide havingamino acid sequence as set forth in SEQ ID NO: 1, and saidpolynucleotide fragment is operably linked to an flower specificpromoter having sequence as set forth in SEQ ID NO: 5; (b) transformingtobacco cells with said host cell by Agrobacterium mediatedtransformation method; and (c) selecting and developing a transgenictobacco plant capable of heterologously expressing a polypeptide havingamino acid sequence as set forth in SEQ ID NO: 6, wherein said tobaccoplant is a male sterile tobacco plant.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic male sterile plants, said methodcomprising: (a) obtaining a transgenic plant biological materialcomprising DNA; (b) carrying out an amplification reaction using primerswhich bind to a DNA sequence as set forth in SEQ ID NO: 7 to generateamplicons; (c) detecting the presence of said amplicons, whereinpresence of said amplicons is indicative of the transgenic plant beingcytoplasmic male sterile.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic male sterile plants as described herein,wherein said primers comprise at least one primer pair comprising aforward, and a reverse primer, wherein both forward primer and reverseprimer do not substantially bind to a region as set forth in SEQ ID NO:3.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic male sterile plants as described herein,wherein said primers comprise at least one primer pair comprising aforward, and a reverse primer, wherein both forward primer and reverseprimer do not substantially bind to a region as set forth in SEQ ID NO:4.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic male sterile plants as described herein,wherein said primers comprise at least one primer pair comprising aforward, and a reverse primer, wherein both forward primer and reverseprimer do not substantially bind to a region as set forth in SEQ ID NO:3 or SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic male sterile plants as described herein,wherein said primers comprise at least one primer pair comprising aforward, and a reverse primer, wherein said forward primer substantiallybinds to a region as set forth in SEQ ID NO: 3, and said reverse primersubstantially binds to a region as set forth in SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic male sterile plants as described herein,wherein said primers comprise at least one primer pair comprising aforward, and a reverse primer, wherein said forward primer substantiallybinds to a region as set forth in SEQ ID NO: 4, and said reverse primersubstantially binds to a region as set forth in SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic male sterile plants as described herein,wherein said transgenic plant is a monocot.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic male sterile plants as described herein,wherein said transgenic plant is a dicot.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic male sterile plants as described herein,wherein said transgenic plant is selected from the group consisting ofkidney bean, lima bean, mung bean, black gram, broad bean, field bean,garden pea, chick pea, black eyed pea, pigeonpea, tobacco, rice, maize,wheat, sorghum, and lentil.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic cytoplasmic male sterile plants asdescribed herein, wherein said transgenic plant is pigeonpea or lentil.

In an embodiment of the present disclosure, there is provided a methodof identification of transgenic cytoplasmic male sterile plants asdescribed herein, wherein said transgenic plant is tobacco.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment comprising a first, and a secondsequence, wherein the first sequence encodes a mitochondrial transitpeptide, and said second sequence encodes a polypeptide having aminoacid sequence as set forth in SEQ ID NO: 1.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as describe herein, wherein said secondsequence is as set forth in SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as describe herein, wherein saidmitochondrial transit peptide is selected from the group consisting ofmitochondrial transit peptide of the cytochrome oxidase subunit IV fromyeast, and COX4 from Saccharomyces cerevisiae (P04037|1-25).

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as describe herein, wherein saidmitochondrial transit peptide amino acid sequence is as set forth in SEQID NO: 2.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as describe herein, wherein saidmitochondrial transit peptide is encoded by SEQ ID NO: 4.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as describe herein, wherein saidpolynucleotide fragment sequence is as set forth in SEQ ID NO: 7.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as describe herein, wherein saidpolynucleotide fragment encodes a polypeptide having amino acid sequenceas set forth in SEQ ID NO: 6.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as described here in, for use ingenerating male sterile plants.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as described here in, for use indetection of male sterile plants comprising a polynucleotide fragment,said polynucleotide fragment comprising a first, and a second sequence,wherein said first sequence encodes a mitochondrial transit peptide,said second sequence encodes a polypeptide having amino acid sequence asset forth in SEQ ID NO: 1.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as described herein, wherein saidsecond sequence is at least 80% similar to SEQ ID NO: 3.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as described herein, wherein saidsecond sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar to SEQID NO: 3.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment as described herein, wherein saidfragment is derived from Cajanus cajan (L.) Millsp. (Pigeonpea).

In an embodiment of the present disclosure, there is provided a methodof inhibiting pollen production in plants, said method comprisingtransforming plant cells with a DNA construct or a recombinant hostcell, said DNA construct or host cell as described herein.

In an embodiment of the present disclosure, there is provided anisolated polynucleotide fragment encoding a protein involved inrestoration of cytoplasmic male sterility in pigeonpea.

In an embodiment of the present disclosure, there is provided a methodof producing synthetic restorers by genetic transformation, said methodcomprising: (a) isolating DNA fragment having at least 90% homology to aDNA fragment encoding a polypeptide having a signal peptide sequence fortranslocation of said polypeptide to mitochondria, wherein saidpolypeptide is involved in restoration of fertility of a male sterileplant, selected from the group consisting of 14 or morepentatricopeptide repeats; and (b) transforming a monocot or dicot plantcell(s) with a DNA construct comprising said DNA fragment from step (a)or recombinant host cell comprising said DNA construct.

In an embodiment of the present disclosure, there is provided a methodof developing hybrid plant in a monocot or dicot plant species bycrossing the transgenic male sterile plant with a plant in which thefertility restoring gene is capable of restoring male sterility, whereinsaid male sterile plant is as described herein.

In an embodiment of the present disclosure, there is provided a methodto modify pollen production of sterile lines which can be used inmonocot or dicot plant species.

In an embodiment of the present disclosure, there is provided a genespecific marker to trace and detect impurity in hybrid seeds ofpigeonpea.

In an embodiment of the present disclosure, there is provided a genespecific marker to detect seed purity of parental lines of pigeonpea.

In an embodiment of the present disclosure there is provided a genespecific marker as described herein, wherein the gene specific markerhaving sequence is as set forth in SEQ ID NO. 7. In another embodiment,the SEQ ID NO. 7 comprises SEQ ID NO: 3. In yet another embodiment, theSEQ ID NO. 7 comprises SEQ ID NO: 4. In further embodiment, the SEQ IDNO. 7 comprises SEQ ID NO. 3 and SEQ ID NO: 4.

In an embodiment of the present disclosure there is provided a genespecific marker as described herein, wherein the gene specific markerencodes for a polypeptide having sequence as set forth in SEQ ID NO: 6.

In an embodiment of the present disclosure, there is provided a methodfor screening a population of plant with a gene specific marker havingsequence as set forth in SEQ ID NO: 7, wherein said gene specific markeris linked to a cytoplasmic male sterility gene in plants. In anotherembodiment, the plant is pigeonpea

In an embodiment of the present disclosure there is provided an isolatedpolynucleotide fragment encoding a protein involved in restoration ofcytoplasmic male sterility in pigeonpea.

In an embodiment of the present disclosure, there is provided a methodof producing synthetic restorers by genetic transformation, said methodcomprising: (a) isolating DNA fragment having at least 90% homology to aDNA fragment encoding a polypeptide having a signal peptide sequence fortranslocation of said polypeptide to mitochondria, wherein saidpolypeptide is involved in restoration of fertility of a male sterileplant, selected from the group consisting of 14 or morepentatricopeptide repeats; and (b) transforming a monocot or dicot plantcell(s) with a DNA construct comprising said DNA fragment from step (a)or recombinant host cell comprising said DNA construct.

In an embodiment of the present disclosure, there is provided a methodof developing hybrid plant in a monocot or dicot plant species bycrossing the transgenic male sterile as described herein, with a plantin which the fertility restoring gene is capable of restoring malesterility.

In an embodiment of the present disclosure, there is provided anisolated DNA encoding a protein involved in restoration of cytoplasmicmale sterility to fertility in plants. In another embodiment, the plantis pigeonpea.

Although the subject matter has been described in considerable detailwith reference to certain preferred embodiments thereof, otherembodiments are possible.

EXAMPLES

The disclosure will now be illustrated with working examples, which isintended to illustrate the working of disclosure and not intended totake restrictively to imply any limitations on the scope of the presentdisclosure. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood to one ofordinary skill in the art to which this disclosure belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice of the disclosed methods and compositions,the exemplary methods, devices and materials are described herein. It isto be understood that this disclosure is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary.

Materials and Methods

Plant material: Seeds of an A₄ cytoplasm (Cajanus cajanifolius)containing CMS line, designated ICPA 2039 was procured from thePigeonpea Breeding Unit of the International Crops Research Institutefor Semi-Arid Tropics (ICRISAT) in Hyderabad, India. The plants weregrown in pots containing autoclaved sand and soil (1:1) mixture, andmaintained in a greenhouse under a 16/8 h light/dark cycle at 25/23° C.with 70-80% relative humidity.

Strains and plasmids: Escherichia coli strains were grown inLuria-Bertani (LB) medium, supplemented with kanamycin (50 μg ml⁻¹) orcarbenicillin (100 μg ml⁻¹) when appropriate. The pET-19b (+) expressionvector and the E. coli strain BL21 (DE3) pLysS (Novagen) were used forprokaryotic expression studies.

Examples

Genomic DNA extraction and genome walking: Total genomic DNA sampleswere prepared from the fresh leaves of one to two week-old seedlingsusing the NucleoSpin Plant II DNA isolation kit (Macherey-Nagel,Germany). Approximately, 500 ng of the genomic DNA was used for genomewalking according to Reddy et al. (Anal. Biochem., 2008, 381, 248-253)to identify the flanking sequences. Contigs from the published pigeonpeamitochondrial genomes (Tutej a et al. DNA Res., 2013, 20, 485-495) ofICPA 2019 (male sterile) and ICPB 2039 (maintainer) were compared insilico. Based on unique rearrangement sites, a 1.291 kb region ofgenomic DNA upstream of the nad7 gene in both sterile and fertile lineswas amplified and cloned using PCR-based directional genome walking witha cDNA sequence specific antisense primer (SEQ ID NO: 8).

RNA isolation: Total RNA was isolated from unopened flower buds andleaves from three-week old plants using Trizol (Invitrogen) and theRNeasy Plant Mini Kit (Qiagen) following the manufacturer's protocol.Total RNA was used for cDNA synthesis using the First-Strand cDNASynthesis Kit (Invitrogen) according to the manufacturer's instructions.The concentration and purity of all RNA samples was tested using aNanoVue plus spectrophotometer (GE health care, USA) at 260/280 nmabsorbance. Only samples whose absorbance ranged from 1.8 to 2.0 wereselected. The integrity of the RNA was confirmed by electrophoresis on1.4% agarose gels.

The cDNAs from ICPA 2039 (both sterile line, and fertile line) and ICPB2039 (male sterile line) were used for the amplification of orf 133 (402bp, SEQ ID NO:46) (using primers having sequence of SEQ ID NO: 9, andSEQ ID NO: 10), and orf 147 (444 bp, SEQ ID NO: 3) (using primers havingsequence of SEQ ID NO: 11, and SEQ ID NO: 12). PCR fragments were usedas templates for Re-PCR with primers (SEQ ID NO: 13, and 14 correspondto forward and reverse primers respectively for amplifying orf133; SEQID NO: 15, and 16 correspond to forward and reverse primers respectivelyfor amplifying orf147) containing restriction sites at the 5′ ends forcloning into a bacterial expression plasmid. The PCR fragments werecloned into the “pJET 2.1 Blunt” plasmid (Thermo) which was confirmed bysequencing. Mini-preparations were used to isolate the recombinantplasmid and the correct orientation of the insert was ascertained byrestriction analysis.

Monitoring E. coli growth rate: Overnight-grown cultures of BL21 (DE3)pLysS (Novagen) harboring pET147, pET(133) or pET32a control plasmidswere obtained by using a single colony to inoculate 1.5 ml of LB mediumsupplemented with carbenicillin and incubated overnight at 37° C. Aflask containing 200 ml of LB medium supplemented with the sameantibiotic was inoculated with 200 μl of the pre-culture and incubatedat 37° C. with shaking. At an OD₆₀₀ of 0.3, the culture was separatedinto two equal subcultures which were induced with 0.5 mM IPTG. Thegrowth of the cultures was monitored on an hourly basis at 600 nm usinga spectrophotometer (Eppendorf Bio Photometer plus).

Vector construction: The 886 bp fragment of the A. thalianaflower-specific AP3 promoter (SEQ ID NO: 5) was amplified using primersAtAP3_Pro_KpnIF (SEQ ID NO: 17) and AtAP3_Pro_NdeIR (SEQ ID NO: 18)followed by cloning in pJET blunt 2.1 plasmid CoxIV (mitochondrialtransit peptide of the cytochrome oxidase subunit IV from yeast) (SEQ IDNO: 4) (Köhler et al., Mol. Gen. Genet., 1997, 227, 369-376). Thepre-sequence was amplified using the TSPF and TSPR primers (SEQ ID NO:19, and 20 respectively) from Saccharomyces cerevisiae cDNA. The Orf147fragment was amplified using primers Orf 147F and Orf 147 Not1R, andsubsequently fused to CoxIV by overlap extension PCR using the primersOE 147F and OE 147R (SEQ ID NO: 21 (forward) and SEQ ID NO: 22(reverse); SEQ ID NO: 23 (forward with restriction site), and SEQ ID NO:24 (reverse with restriction site)). The PCR amplified AP3 promoterfragment (KpnI, NdeI) and Cox-orf147 fusion fragment (NdeI, NotI) weretogether sub-cloned into a modified pL12R34H plasmid at the KpnI, NotIsite, then into pMDC100 followed by mobilization into Agrobacteriumtumefaciens strain C58 for plant transformation studies in Arabidopsisand tobacco.

Plant transformation: Arabidopsis thaliana seeds (Col-1) were germinatedin 4-cm pots and maintained in culture room conditions until thefour-leaf stage. This was followed by transfer to a glasshouse wherethey were irrigated every four days until appearance of theinflorescences. Once the inflorescences reached about five cm, plantswere transformed with A. tumefaciens suspension harboring the genes ofinterest using the floral dip protocol (Clough et al., Plant J., 1998,16, 735-743), with inoculations repeated twice at three-day intervals.The seeds were collected at maturity. Tobacco (Nicotiana tabacum L.,var. Xanthi) seedlings were grown under controlled sterile-environmentconditions for two weeks followed by Agrobacterium-mediatedtransformation using the standard leaf disc method (Sunkara et al.,Appl. Biochem. Biotechnol., 2013, 172, 325-339. DOI:10.1007/s12010-013-0482-x). Transgenic plants were grown in potscontaining autoclaved sand and soil (1:1) in a containment greenhouseuntil flowering and seed formation set in. A 16/8 h light/dark cycle at23/20° C. with 65-70% relative humidity was used for Arabidopsis and25/23° C. with 70-80% relative humidity for tobacco.

Molecular characterization of transgenic plants: Genomic DNA fromkanamycin resistant N. tabacum and A. thaliana plants was isolated usingthe NucleoSpin Plant II DNA isolation kit and subjected to PCR usingorf147 specific primers. PCR conditions included an initial denaturationcycle of 5 min at 94° C., followed by 35 cycles of denaturation for 30 sat 94° C., annealing for 1 min at 58° C. with an extension for 1 min at72° C. and a final extension for 10 min at 72° C.

Candidate gene selection and primer design: Genome walking in malesterile and fertile lines as well as cDNA amplification was carried outusing specific primers. Seven candidate genes were selected for qRT-PCRanalysis, which included genes associated with anther biogenesis, whichhave key roles in normal tapetal function and viable pollen production.These were Defective in tapetal development and function (TDF1/MYB35);Aborted Microspore (AMS) (forward primer SEQ ID NO: 27; reverse primerSEQ ID NO: 28); Dysfunctional Tapetum1 (DYT1) (forward primer SEQ ID NO:25; reverse primer SEQ ID NO: 26), and Male Sterility 1 (MS1). The otherthree genes which were from the lignin biosynthetic pathway viz., 4CL (4Coumarate:CoAligase)(forward primer SEQ ID NO: 39; reverse primer SEQ IDNO: 40), CCoAOMT (Caffeoyl CoA O-Methylransferase) (SEQ ID NO: 37, andSEQ ID NO: 38), and C3H (Cinnamic acid 3-hydroxylase) (SEQ ID NO: 35,and SEQ ID NO: 36) were selected from the Arabidopsis database (TIAR)and used for qRT-PCR analysis. Three reference genes SAND (SEQ ID NO:29, and SEQ ID NO: 30), TIP41 (SEQ ID NO: 31, and SEQ ID NO: 32) and UNK(SEQ ID NO: 33, and SEQ ID NO: 34), showing highly stable expression(Czechowski et al., Plant Physiol., 2005, 139, 5-17. doi:10.1104/pp.105.063743) were selected as reference genes for this study. Theretrieved A. thaliana sequences were used to design PCR primers usingPrimer 3 Plus software. The primers had a GC content of 50%, a length of22 nucleotides and an expected product size of 80-150 base pairs.

Quantitative real time PCR analysis: All qRT-PCR reactions were carriedout in a Realplex Real-Time PCR system (Eppendorf, Germany) using SYBRGreen in 96 well optical reaction plates (Axygen, Union City, Calif.,USA) sealed with ultra-clear sealing film (Platemax). The PCR reactionwas performed in a total volume of 10 μl containing 1 μl of RNA (100ng), 400 nM of each primer, 5 μl of 2× one step SYBR RT-PCR buffer 4(Takara, Japan) and 0.4 μl of prime script one step Enzyme Mix 2(Takara, Japan) made up to 10 μl with RNase-free H₂O. The qRT-PCRcycling conditions were as follows: 42° C. for 5 min and 95° C. for 10 s(reverse transcription) followed by 40 cycles of 15 s at 95° C., 15 s at62° C. with fluorescent signal recording and 15 s at 72° C. The meltingcurve analysis was included after 40 cycles to verify the primerspecificity by heating from 58° C. to 95° C. with fluorescence measuredwithin 20 min. No-template controls were included for each of the primercombinations. All the samples were collected from the three independentplants and each sample was tested in three technical replicates. The rawquantification cycle (Cq) values of each gene were taken as the inputdata to estimate relative and average expression of the candidate geneusing qBase plus software (ver: 2.4; Biogazelle, Belgium) (Hellemans etal., genome Biol., 2007, 8: R19. doi: 10.1186/gb-2007-8-2-r19).

Histochemical studies: The lignin content in the A. thaliana and N.tabacum flower buds was histochemically analyzed usingphloroglucinol-HCl staining. The flowers were fixed in FAA solutionovernight and decolorized by using ethanol 25-85% series. These weresubsequently stained with 2% (w/v) phloroglucinol in 92.5% ethanol for 1h at room temperature, following which the tissues were mounted with18.5% (v/v) HCl. The red coloration was monitored immediately using aLeica M125 microscope (Leica Microsystems; Bannockburn; IL, USA).

Results

CMS (Cytoplasmic male sterility) specific sequence in male sterilecytoplasm: To explore the CMS causing genes, previously reportedrearrangement sites unique to ICPA 2039 (Tuteja et al., DNA Res., 2013,20, 485-495), were initially compared in pigeonpea mitotypes.Interestingly, upon comparing the flanking sequences of the nad7 gene inthe male sterile line with those in the maintainer line, a variablefragment was found to be located 5′ to the nad7 subunit of complex I(the main dehydrogenase of the mitochondrial respiratory chain) in themale sterile line of pigeonpea. While genome walking revealed sequencevariations in the 5′ upstream region of nad7 of male fertile and sterilelines, the coding as well as the 3′ regions were observed to beidentical (FIG. 1A). Sequence divergence was observed in the upstreamregion starting from −259 bp of the nad7 initiation site. In FIG. 1A, Mrefers to protein marker ladder; S refers to male sterile line, while Frefers to fertile line.

ORF prediction and expression validation by RT-PCR: Predictive analysesof nucleotide sequences of the nad7 region in the fertile parent, ICPB2039, and in the ICPA 2039-CMS line revealed two ORFs based on athreshold of 85 amino acids, with a reasonably high level ofvariability. Reverse transcription-PCR (RT-PCR) analysis using differentsets of primers resulted in amplification of various regions includingORF sequences upstream of the nad7 gene. cDNAs of both fertile andsterile pigeonpea lines amplified a 402 bp fragment referred to asorf133 (FIG. 1B), revealing three amino acid differences between thefertile and sterile lines (data not shown). In FIGS. 1B, and 1C, Srefers to male sterile plant, F refers to fertile plant, R refers torestorer plant, while H refers to hybrid plant. However, primer set (SEQID NO: 11, and 12) resulted in the amplification of 444 bp fragmentspecific only to the male sterile line (ICPA 2039), hereby called asorf147 (SEQ ID NO: 3) (FIG. 1C). A database search for the similarity ofthe orf147 gene fragment from the sterile line to known ORFs in thedatabase using BLASTX (www.ncbi.nlm.nih.gov/BLAST) detected nosignificant sequence homology.

However, its deduced amino acid sequence showed partial homology to“orf124” of Beta vulgaris subsp. maritime genotype male-sterile Emitochondrion (accession #FQ014226.1).

orf147 transcription is polycistronic: RT-PCR carried out using primercombinations specific to different internal regions viz. orf147F (SEQIDNO:11)/nad7PGER (SEQID NO: 41), orf133F (SEQ ID NO: 9)/nad7P1R (SEQIDNO:42), and orf133F (SEQID NO: 9)/nad7PGER (SEQID NO: 41) resulted inamplification of 1,741 bp, 1,327 bp, and 2,143 bp, respectively. Theseoverlapping amplicons indicated the presence of a single 2,455 bppolycistronic transcript in the mitochondria from the sterile cytoplasmencompassing orf133, orf147 and nad7 and spacer sequences (FIG. 2A) Incontrast, the fertile maintainer line did not show any amplificationwith any of these primer sets, indicating the monocistronic nature ofthe nad7 transcript (FIG. 2 B-C). (in FIGS. 2B & C, S refers to sterileplant, F refers to fertile plant, R refers to restorer plant, while Hrefers to hybrid plant). Nevertheless, orf133 amplification withgene-specific primers (orf133-F/orf133-R) in the maintainer lineindicated its existence as a separate transcript (FIG. 1B).

These results were further confirmed by transcription start site (TSS)identification in the male sterile and fertile pigeonpea lines carriedout using two different sets of RACE Primers. While PCR with primersRACE F1 (SEQID NO:43) and RACE R1 (SEQID NO:44) following cDNAcircularization, resulted in amplification in both male sterile(ICPA2039) and maintainer (ICPB2039) lines, the primers RACE F1 and RACER2 (SEQID NO:45) showed amplification with the male sterile line (ICPA2039) only, thereby indicating absence of this region in the cDNAtranscript (FIG. 3A). For the male sterile line (ICPA 2039), thesequence results using RACE F1 and RACE R2 (referred to as ICPA“transcript 1”) identified a T residue located 1,681 bp upstream to theNad7 start codon as the TSS (FIG. 3A), while the product of RACE F1 andRACE R1 (referred to as “transcript 2”) showed a G at 656 bp upstream ofthe nad7A start codon as the other functional TSS (FIG. 3B), with“orf147” in common. For the male fertile maintainer line (ICPB 2039),the sequence analysis revealed the TSS at T (−556) with primers RACE F1and RACE R1 (FIG. 3C). These results confirmed that the orf147transcripts in the male sterile line existed with more than one cistron,while the male fertile line had a monocistronic transcript (FIG. 3D).

RNA editing and secondary structure of orf147: The cDNA sequence of the5′ upstream region of the nad7 gene in both male sterile and fertilelines were compared with their respective mitochondrial genome sequencesand the genome-walked PCR products. This identified a differential RNAediting pattern in the sequenced clones for each line. Severalconsistent edited sites among independent clones were detected for eachline. The male sterile line (ICPA 2039) exhibited 10 edited changes inthis region, in contrast to 22 observed in the maintainer line. Therewere four edited events in the CMS line in orf133: a glycine residue atthe 59 aa residue position was edited to serine; glutamine at 119 wasedited to leucine; serine at 124 was changed to arginine and isoleucineat position 125 was edited to lysine. However, there was no edited eventobserved in orf147.

The secondary structure of the orf147 transcripts of the CMS linerevealed a perfect hairpin loop structure at the 5 end (FIG. 4A). Insilico analysis using a homology-based modeling program (www.expasy.org)suggested that the product of orf147 does not contain any trans-membranedomain and might be a soluble protein (FIG. 4B).

orf147 encodes a cytotoxic peptide: To examine the function of theorf147 and orf133 transcripts, their coding sequences were cloned intothe expression region of the PET32a vector, followed by IPTG-inducedexpression in E. coli. While the growth curve analysis of orf133expressing cells showed no apparent effect on growth upon IPTGinduction, induction of orf147 expressing cells resulted in cytotoxicityto the E. coli cells (FIG. 5A, B) (in FIG. 5A, pET32a UI refers touninduced control vector, pER32a I refers to induced control vector,Orf147 pET32a UI refers to uninduced vector comprising orf147, andOrf147A pER32a I refers to induced vector comprising orf147) (in FIG.5B, pET32a UI refers to uninduced control vector, pER32a I refers toinduced control vector, pER32a ORF133UI refers to uninduced vectorcomprising orf133, and pET32a ORF133 I refers to induced vectorcomprising orf133). The induction levels of orf147 (with His tag), andorf133 (with His tag) were also visualized by resolving the fusionproteins on 12% SDS-PAGE gel. As seen in FIG. 5A, lane 1 depicts pET 32auninduced; lane 2 depicts pET 32a induced; lane 3 depicts orf147 pET 32auninduced; and lane 4 depicts ORF147 pET 32a induced. In FIG. 5B, lane 1depicts pET 32a uninduced; lane 2 depicts pET 32a induced; lane 3depicts ORF133 pET 32a uninduced; and lane 4 depicts orf133 pET 32ainduced.

Bacterial cell growth was also ascertained by evaluating colony growthof transformed bacteria in agar plates upon 0.5 mM IPTG induction andgrowth monitored on an hourly basis at 600 nm using a spectrophotometer.As seen in FIG. 5C, pET32a control without induction (vector alone), andpET32a Orf133 without induction show no decrease in cell density/growth.Similarly, pET32a Orf147 without induction also does not show any celldensity/growth defect compared to control. pET32a Orf133 when induced,did not show any growth defect when compared to induced control pET32a(vector alone). However, pET32a Orf147 upon induction showed a severegrowth defect compared to induced control pET32a (vector alone), furthersupporting the conclusion that expression of Orf147 is toxic totransformed E. coli cells.

Expression of orf147 in Arabidopsis and tobacco results in malesterility: Transformation with the AP3::CoxIV-Orf147 gene cassette (FIG.6A) carrying the cox/V-orf147 gene fusion driven by the AtAP3 promoter,resulted in 24 primary transgenic events in Arabidopsis, and over 20 intobacco. These were grown to maturity and T1 seeds collected. Vegetativegrowth of the transgenic plants (ie, growth rate and plant morphology)was uniform and similar to that of the untransformed counterparts inboth species.

About 80% of the T1 progeny from the selected 12 independent Arabidopsisevents at flowering, exhibited the semi-sterile or sterile phenotype,resulting in poor seed setting when compared to the wild type (WT) (FIG.6 B-C) (FIG. 6B depicts male sterile transgenic Arabidopsis plantshowing normal growth and development; FIG. 6C depicts wild type plantwith primary branches showing normal siliques). At the dehiscent stage,the sterile transgenic events did not produce any pollen grains andnormal siliques. The sterile plants had flowers with smaller sepals andpetals than their WT counterparts, with a protruding pistil andshortened stamen filaments with impaired anther dehiscence. Thesemi-sterile plants bore two kinds of siliques, one shorter and with noor fewer seeds than the WT and the other normal siliques like the WT.The male sterile plants had very short siliques with no seeds (FIG. 6D-F) (FIG. 6D depicts male sterile transgenic plant with short siliquesindicating no developing seeds (top); FIG. 6E depicts front view ofnormal mature flowers of WT (inset shows normal anther dehiscence); andFIG. 6F depicts flowers of male sterile line revealed fused carpels,protruding pistil and short filaments (inset non-dehiscent anther in thetransgenic flower)).

Similarly, out of 20 primary transgenic events of tobacco, fourconfirmed positive events showed complete male sterility. The flowers ofmale sterile transgenic tobacco plants expressing orf147 were againrelatively smaller with shortened filaments and either produced verysmall fruits exhibiting partial sterility or had detached collapsedcapsules in the completely sterile plants (FIG. 6 G-J) (FIG. 6G depictsflower size, color and structure in the WT tobacco plant; FIG. 6Hdepicts flowers of male sterile tobacco plants having anthers below thestigma; FIG. 6I depicts Top: Seed capsules from N. tabacum (WT); Bottom:Sterile progeny; and FIG. 6J depicts Seed capsules of WT plants (Left),collapsed and detached seed capsules in partially sterile transgenicphenotypes (Inset) Floral branches from wild type (WT)).

The qRT-PCR analysis of several selected transgenic Arabidopsis andtobacco plants revealed variation in the orf147 transcript levels, withmale sterile phenotypes showing strong orf147 expression (FIG. 7 A, B).In FIG. 7A, ICPA2039 refers to a male sterile pigeonpea plant showinghigh relative expression of orf147, while ICPB 2039 is a fertilepigeonpea plant showing negligible orf147 expression. These data showthat orf147 gene expression is associated and limited to male sterileplants only in pigeonpea. In FIG. 7B, the left column depicts orf147expression in transgenic Arabidopsis which are fully male sterile, whilethe right column depicts orgf147 expression in transgenic tobacco, whichare partially male sterile, showing that heterologous expression oforf147 in mitochondria of transgenic Arabidopsis or tobacco results inmale sterility.

Expression of anther development related-genes: To detect the expressionof anther development-related genes that act after tapetalspecification, quantitative RT-PCR analysis for key genes involved inanother development was carried out in male sterile and fertilepigeonpea lines (ICPA 2039 and ICPB 2039 respectively) and in transgenicArabidopsis plants along with their WT. Interestingly, in the CMSpigeonpea line, while the transcripts of Defective in tapetaldevelopment and function (TDF1/MYB35), Dysfunctional tapetum1 (DYT1),and Male sterility1 (MS1) were significantly down-regulated comparedwith those in the fertile maintainer line, an increased accumulation oftranscripts of the Aborted microspore (AMS) gene was observed (FIG. 8A).

Similarly, the ectopic expression of orf147 in Arabidopsis transgenicplants resulted in significant down-regulation of the transcripts of AMSand MS1 that are required for normal tapetal function and pollen walldevelopment (FIG. 8B). This data suggested that mitochondrial expressionof orf147 from the pigeonpea male sterile line induces male sterility intransgenic Arabidopsis plants, possibly by regulating thetranscriptional expression of key genes specific to another development.

Male sterile anthers have reduced endothecium secondary wallliginifcation: Investigation of lignification patterns usingphloroglucinol staining of anthers of both wild-type and transgenic malesterile flowers of Arabidopsis and tobacco revealed a high degree ofphloroglucinol stain accumulation in the WT anthers at all stages ofdevelopment, when compared to the anthers of the male sterile transgenicline (FIG. 9A-F).

Further, these observations correlated well with the gene expressionprofiles of key genes that are involved in lignin biosynthesis like4CL(4 coumarate:CoAligase), CCoAOMT (caffeoyl CoA O-methyltransferase),and C3H (cinnamic acid 3-hydroxylase). Clearly, these genes wereexpressed at significantly lower levels in the flowers of the malesterile line when compared to the WT. The relative expression of 4CL,C3H, CCoAOMT were 0.85, 0.55 and 0.8 in male sterile plants, as comparedto 1.18, 1.7 and 1.25 in the WT plants (FIG. 9G).

CONCLUSIONS

The transcription and translation patterns of the predicted ORFs in thecontigs spanning a 10 kb region situated upstream and downstream of theknown nad7 gene were comprehensively evaluated in the mitochondrialgenomes of pigeonpea genotypes ICPA 2039 and ICPB 2039. In the presentdisclosure, there is disclosed a 444 bp long unique CMS-associated novelorf147 (SEQ ID NO: 3) detected upstream of and co-transcribing with theknown nad7 gene in the mitochondrial genome of pigeonpea male sterileICPA 2039 cytoplasm is very likely to be responsible for mitochondrialdysfunction. There is no indication of the novel transcript resulting ina reduced/loss or gain of function change in the nad7 gene per seindicating no apparent bearing on the oxidative phosphorylation.

The results of the present disclosure contradict and diverge from arecent report (Sinha et al., Plant Genome, 2015, Genome 8: 1-12. doi:10.3835/plantgenome2014.11.0084), where a frame-shift mutation in thenad7 gene and the resulting disordered predicted protein structure wasreported to be the cause of CMS in pigeonpea. However, these claims onthe aberration in the nad7 gene, prediction of a chimeric ORF as thesterility factor causing conformational changes in the secondary andtertiary nad7 protein structure lacked experimental evidences. Besides,the cDNA clones of nad7 from CMS and fertile pigeonpea lines did notreveal any differences in the sequence and expression of nad7 and/ordeduced protein structures in our study. The present data on thestructural and functional variations in the male sterile and fertilelines, comprehensive characterization of the causal ORF, and itsfunctional validation in both prokaryotic and eukaryotic biologicalsystems clearly disagree with the conformational differences andinterpretations drawn by Sinha et al., 2015 and calls for a thoroughmethodological review of the ORF and protein prediction analyses inSinha et al., 2015.

The secondary structure of orf147 transcripts of the male sterilepigeonpea line reveal a perfect hairpin loop structure at the 5 end thatwas previously suggested to provide stability to the malesterility-associated transcripts in CMS.

Orf147 is a soluble protein cytotoxic to E. coli and its recombinanttransgene leads to male sterility in two tested model plant species. Toeliminate the possibility of the observed toxic effects resulting fromoverloading of the protein synthesis machinery of overexpressedheterologous ORF147 proteins in E. coli cells, a similar expressionstudy was carried out with orf133 (SEQ ID NO:46), another existingupstream ORF in the CMS line, whose accumulation had no adverse effecton the growth of E. coli. This suggests that such a characteristic oforf147 might also affect the development of floral organs. In previousreports, CMS-associated proteins such as PCF in Petunia, ORF125 inKosena radish (Raphanus sativus cv. Kosena; Nivison et al., Plant Cell,1989, 1, 1121-1130; Iwabuchi et al., Plant Mol. Biol., 199, 39, 183-188)and expression of orf 79 in BT-type CMS rice have been shown to becytotoxic (Duroc et al., Biochimie, 2005, 87 1089-1100; Wang et al.,Plant Cell, 2006, 18, 676-687).

Functional validation of the effect of the orf147 gene on male sterilityusing tapetum-specific expression of the orf147 gene in A. thaliana andN. tabacum transgenic plants in the present disclosure resulted inpartial to complete male sterility, thereby suggesting that the encodedcytotoxic protein results in disruption of the development of malesporophytic and/or gametophytic cells.

The tissue-specific expression of orf147 not only disturbs thedifferentiation of stamens, but also affected the development of petalsin the transgenic Arabidopsis plants in the present disclosure, howeverno morphological differences were observed in the tobacco transgenicplants. This could possibly be due to a relatively weaker expression inthe flowers under the influence of a heterologous Arabidopsis promoterused for transformation experiments. The male sterile or semi-sterileArabidopsis as well as tobacco transgenic plants formed shortened stamenfilaments. Interestingly, there were no notable differences in thevegetative growth of the transgenic plants and their wild typecounterparts in both the tested plant species, which could be attributedto the specific interaction of CMS-associated genes with floral organs(Jing et al. J. Exp. Bot., 2012, 63, 1285-1295). The male steriletransgenic phenotype in both Arabidopsis and tobacco was heritable andstrong orf147 expression in the T₁ progeny indicated completepenetrance, an important finding in terms of research on the CMSmechanism in this pulse crop.

Overall, the present disclosure provides a novel isolated polynucleotidefragment of SEQ ID NO: 3 from pigeonpea, spatial heterologous expressionof which in the mitochondria causes cytoplasmic male sterility (CMS).The disclosure further provides DNA constructs, and reagents, which canbe used to generate transgenic plants to design CMS lines in crops withno previously available male sterile lines. This heritable and traceablefragment can be used to develop and maintain new hybrids acrossdifferent plant species, and significantly reduces the time spent inidentifying naturally occurring cytoplasmic male sterility factors.

We claim:
 1. A DNA construct comprising a polynucleotide fragment, saidpolynucleotide fragment comprising a first sequence and a secondsequence, wherein said first sequence encodes a mitochondrial transitpeptide, said second sequence encodes a polypeptide having an amino acidsequence as set forth in SEQ ID NO: 1, and said polynucleotide fragmentis operably linked to a flower or stamen specific promoter.
 2. The DNAconstruct as claimed in claim 1, wherein said mitochondrial transitpeptide is selected from the group consisting of a mitochondrial transitpeptide of the cytochrome oxidase subunit IV from yeast, and a COX4 fromSaccharomyces cerevisiae (P04037|1-25).
 3. The DNA construct as claimedin claim 2, wherein said mitochondrial transit peptide amino acidsequence is as set forth in SEQ ID NO: 2, and is fused in-frame at the5′ end of said second sequence.
 4. The DNA construct as claimed in claim1, wherein said polypeptide is encoded by SEQ ID NO:
 3. 5. The DNAconstruct as claimed in claim 3, wherein said mitochondrial transitpeptide is encoded by SEQ ID NO:
 4. 6. The DNA construct as claimed inclaim 1, wherein said flower or stamen specific promoter is selectedfrom the group consisting of an AP3 promoter for floral expression fromArabidopsis, an AP3 promoter for floral expression from Tomato, an AP3promoter from Coffea arabica, a TA29 promoter for tapetum-specificexpression from Lycopersicon esculentum, and a TA29 promoter fromtobacco for tapetum-specific expression.
 7. The DNA construct as claimedin claim 6, wherein said flower or stamen specific promoter sequence isas set forth in SEQ ID NO:
 5. 8. A male sterile plant harboring in itsgenome a DNA construct as claimed in claim
 1. 9. The plant as claimed inclaim 8, wherein said plant is a dicot or a monocot.
 10. An isolatedpolynucleotide fragment comprising a first sequence and a secondsequence, wherein said first sequence encodes a mitochondrial transitpeptide, and said second sequence encodes a polypeptide having an aminoacid sequence as set forth in SEQ ID NO:
 1. 11. The isolatedpolynucleotide fragment as claimed in claim 10, wherein saidmitochondrial transit peptide amino acid sequence is as set forth in SEQID NO:
 2. 12. The isolated polynucleotide fragment as claimed in claim11, wherein said first sequence is as set forth in SEQ ID NO: 4, andsaid second sequence is as set forth in SEQ ID NO:
 3. 13. The isolatedpolynucleotide fragment as claimed in claim 10, encoding a polypeptidehaving an amino acid sequence as set forth in SEQ ID NO:
 6. 14. Theisolated polynucleotide fragment as claimed in claim 13 having asequence as set forth in SEQ ID NO:
 7. 15. The isolated polynucleotidefragment as claimed in claim 10, wherein said fragment is derived fromCajanus cajan (L.) Millsp. (Pigeonpea).